Alpha-amylase synthesis in Bacillus subtilis 168 is activated at the end of exponential growth, is apparently not inducible, and is repressed by more-readily-metabolized carbon sources, such as glucose. Temporal activation and catabolite repression of amylase synthesis are distinct, genetically-separable events. We have localized the cis-acting regulatory DNA sequences necessary for proper regulation of amylase synthesis to the amyR region at the 5' end of the amylase gene. We propose experiments to define the DNA sequences within amyR which are involved in amylase regulation and to identify trans-acting factors which interact with these regulatory sites. Specifically, we propose a series of experiments designed to dissect the functional domains of the amyR region using targeted mutagenesis. In addition, we propose to identify and characterize trans-acting regulatory elements through a program combining mutagenesis with studies of physical interactions between trans-regulatory factors and the functional sites within amyR. Elucidation of the mechanism of amylase regulation resulting from these experiments will provide insights into the regulatory mechanisms governing the expression of other postexponentially-expressed genes in B. subtilis.